Methods using polarimetry, colorimetry, reductimetry and a variety of chromatographies have been developed as methods for quantitative analysis of sugars such as sucrose and glucose. These methods, however, are all poorly specific to sugars and hence have poor accuracy. Among these methods, the polarimetry is simple in manipulation, but is largely affected by the temperature during the manipulation. Therefore, the polarimetry is not suitable as a method for simple quantification of sugars at home, etc. for ordinary people.
Recently, various types of biosensors utilizing the specific catalytic action of enzymes have been under development.
The following will describe a method of glucose quantification as one example of the method of quantifying a substrate contained in a sample.
As an electrochemical method of glucose quantification, a method using glucose oxidase (EC 1.1.3.4: hereinafter abbreviated to GOD) as an enzyme and an oxygen electrode or a hydrogen peroxide electrode is generally well-known (see “Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).
GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron mediator. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction process by GOD. The decreased amount of oxygen is measured by the oxygen electrode, or the increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. Since the decreased amount of oxygen and the increased amount of hydrogen peroxide are proportional to the content of glucose in the sample, glucose quantification is possible based on the decreased amount of oxygen or the increased amount of hydrogen peroxide.
The above method utilizes the specificity of the enzyme reaction to enable accurate quantification of glucose in the sample. However, as speculated from the reaction process, there is a drawback that the measurement results are largely affected by the oxygen concentration of the sample, and if the oxygen is absent in the sample, the measurement is infeasible.
Under such circumstances, glucose sensors of new type have been developed which use as the electron mediator potassium ferricyanide, an organic compound or a metal complex such as a ferrocene derivative and a quinone derivative without using oxygen as the electron mediator. In the sensors of this type, reduced form electron mediator resulting from the enzyme reaction is oxidized on a working electrode, and the concentration of glucose contained in the sample can be determined based on the amount of this oxidation current. At this time, on a counter electrode, a reaction in which oxidized form electron mediator is reduced to produce reduced form electron mediator proceeds. With the use of such an organic compound or metal complex as the electron mediator in place of oxygen, it is possible to form a reagent layer while a known amount of GOD and the electron mediator are carried in a stable state and a precise manner on the electrode, so that accurate quantification of glucose is possible without being affected by the oxygen concentration of the sample. It is also possible to integrate the reagent layer containing the enzyme and electron mediator, in an almost dry state, with an electrode system, and hence disposable glucose sensors based on this technique have recently been receiving a lot of attention. A typical example thereof is a biosensor disclosed in Japanese Patent Publication No. 2517153. In such a disposable glucose sensor, it is possible to measure glucose concentration easily with a measurement device by simply introducing a sample into the sensor connected detachably to the measurement device.
In the measurement made by such a glucose sensor, it is possible, with the use of a sample whose amount is in the order of several μl, to determine substrate concentration in the sample readily. However, in recent years, it is anxiously desired in various fields to develop biosensors that enable measurements with the use of a sample in a further smaller amount of about not more than 1 μl. The conventional electrochemical glucose sensors have an electrode system that is arranged on one plane in most cases. When the amount of sample is extremely small, such an arrangement increases the resistance to electric charge transfer between the electrodes, mainly ion transfer, possibly resulting in variations in measurement results.
Thus, there is proposed a biosensor having a working electrode and a counter electrode that are arranged so as to be opposed to each other (Japanese Laid-Open Patent Publication No. Hei 11-350293). In this type of sensor, the opposite arrangement of the working electrode and counter electrode facilitates ion transfer between the working electrode and the counter electrode, and for this reason and other reasons, this type of sensor is capable of quantifying a substrate such as glucose contained in a sample with higher accuracy and higher sensitivity than the conventional biosensors having an electrode system arranged on one plane.
Since it is requested recently to further reduce the amount of sample necessary for measurement, there is a need to realize a biosensor having a further higher sensitivity that enables measurements with the use of a further smaller amount of sample.
In view of the above problems, an object of the present invention is to provide a highly sensitive biosensor that needs a smaller amount of sample for measurement.